2.1.4 Results and discussion
2.1.4.2 Antigen presentation
2D Alkyne 3D Alkyne
2D Ep ox y 3D Ep ox y
3D Ma l
Ne utr av id in Str ep ta vi di n
0 100 200 300 400 500 1000600 2000
Background noise
FU
2D Alkyne 3D Alkyne
2D Ep ox y 3D Ep ox y
3D Ma l
Ne utr av id in Str ep ta vi di n
0 1 2 3 4
CV
CV = coefficient of variance, FU = fluorescent units
Figure 2.6: Fluorescence background intensities of different slide coatings Background measured with buffer blank
Immobilization strategy
Based on the results of our background intensity level test, three different immobilization strategies, needed for each particular surface functionalization, were investigated. As il-lustrated in figure 2.7, a random immobilization on 2D epoxysilan slides (A), a surface coupling on a 2D alkyne slide with an azide modified PSA (B) as well as the connection of the protein to a 2D streptavidin surface via a spacer (C) were compared.
Immobilization on epoxysilanized slides
An immobilization on epoxysilanized slides does not require any protein modification as the protein is covalently linked to the surface via free and deprotonated amino functions, but also to some extent via other nucleophilic thiol and hydroxyl groups.
Click immobilization
For the utilization of alkyne slides, an azide moiety had to be coupled to PSA prior to immobilization. This was achieved by the coupling of NHS-PEG4-N3 to free amine groups of the PSA. To click the azide-modified PSA onto an alkyne functionalized surface required
Random immobilization
epoxy streptavidin alkyne
Surface functionalization
Oriented immobilization
Modification:
Y Y Y Y Y
Y Y Y Y Y Y Y Y YPSA NH2OH HS
PSA
Glyc.
Glyc.
PSA N3 Enyzme
+ UDP-Gal-N3
PSA NH2
Glyc.
+ NHS-PEG-Biotin PSA Glyc.
Biotin
PSA Glyc.
Biotin
PSA N3
+ Cu
PSA Glyc.
PSA
Random immobilization
epoxy streptavidin alkyne
Surface functionalization
Oriented immobilization
Modification:
Y Y Y Y Y
Y Y Y Y Y Y Y Y YPSA NH2OH HS
PSA
Glyc.
Glyc.
PSA N3 Enyzme
+ UDP-Gal-N3
PSA NH2
Glyc.
+ NHS-PEG-Biotin PSA Glyc.
Biotin
PSA Glyc.
Biotin
PSA N3
+ Cu
PSA Glyc.
PSA
Surface immobilizationModificationOrientation
Random immobilization
epoxy streptavidin alkyne
Surface functionalization
Oriented immobilization
Modification:
Y Y Y Y Y
Y Y Y Y Y Y Y Y YPSA NH2OH HS
PSA
Glyc.
Glyc.
PSA N3 Enyzme
+ UDP-Gal-N3
PSA NH2
Glyc.
+ NHS-PEG-Biotin PSA Glyc.
Biotin
PSA Glyc.
Biotin
PSA N3
+ Cu
PSA Glyc.
PSA
Random immobilization
epoxy streptavidin alkyne
Surface functionalization
Oriented immobilization
Modification:
Y Y Y Y Y
Y Y Y Y Y Y Y Y YPSA NH2OH HS
PSA
Glyc.
Glyc.
PSA N3 Enyzme
+ UDP-Gal-N3
PSA NH2
Glyc.
+ NHS-PEG-Biotin PSA Glyc.
Biotin
PSA Glyc.
Biotin
PSA N3
+ Cu
PSA Glyc.
PSA
random random via azide
unmodified
Random immobilization
epoxy streptavidin alkyne
Surface functionalization
Oriented immobilization
Modification:
Y Y Y Y Y
Y Y Y Y Y Y Y Y YPSA NH2OH HS
PSA
Glyc.
Glyc.
PSA N3 Enyzme
+ UDP-Gal-N3
PSA NH2
Glyc.
+ NHS-PEG-Biotin PSA Glyc.
Biotin
PSA Glyc.
Biotin
PSA N3
+ Cu
PSA Glyc.
PSA
Random immobilization
epoxy streptavidin alkyne
Surface functionalization
Oriented immobilization
Modification:
Y Y Y Y Y
Y Y Y Y Y Y Y Y YPSA NH2OH HS
PSA
Glyc.
Glyc.
PSA N3 Enyzme
+ UDP-Gal-N3
PSA NH2
Glyc.
+ NHS-PEG-Biotin PSA Glyc.
Biotin
PSA Glyc.
Biotin
PSA N3
+ Cu
PSA Glyc.
PSA
Random immobilization
epoxy streptavidin alkyne
Surface functionalization
Oriented immobilization
Modification:
Y Y Y Y Y
Y Y Y Y Y Y Y Y YPSA NH2OH HS
PSA
Glyc.
Glyc.
PSA N3 Enyzme
+ UDP-Gal-N3
PSA NH2
Glyc.
+ NHS-PEG-Biotin PSA Glyc.
Biotin
PSA Glyc.
Biotin
PSA N3
+ Cu
PSA Glyc.
PSA
random + spacer
A B C
Random immobilization
epoxy streptavidin alkyne
Surface functionalization
Oriented immobilization
Modification:
Y Y Y Y Y
Y Y Y Y Y Y Y Y YPSA NH2OH HS
PSA
Glyc.
Glyc.
PSA N3 Enyzme
+ UDP-Gal-N3
PSA NH2
Glyc.
+ NHS-PEG-Biotin PSA Glyc.
Biotin
PSA Glyc.
Biotin
PSA N3
+ Cu
PSA Glyc.
PSA PSA
Glyc.
PSA N3 +NHS-PEG4-N3
Glyc.
Glyc. = glycosylation, N3 = azide moiety Figure 2.7: Immobilization strategies
A - Immobilization of unmodified PSA on 2D-epoxysilanzied slide.
B - Azide modification and following ’click’ immobilization on alkyne surface.
C- PSA PEGylation via NHS chemistry and attachment to streptavidin-coated surface.
copper as catalyst. Copper is known to potentially denature proteins, so it had to be tested if PSA would remain recognizable when employed. Surface staining of attached proteins using SyproRuby revealed that PSA could be immobilized via the explained route also indi-cating that the modification was successful (data not shown). However, the gained results shown in figure 2.8 A pointed out that PSA degraded under the influence of copper, as it could not be recognized by the detection antibody anymore. This effect was independent of the copper concentration. An immobilization was observed if no cooper was added, but it can be assumed that this was mainly due to an unspecific interaction - as shown in figure 2.8 B, PSA could be attached to the 2D alkyne surface without any modification, too.
A B
C D
28 kDa 33
42-39
8 9 10 11 12 13 14
0 20 40 60 80 100 120
Ret. time [min]
Abs. 280nm normalized to max. [%]
PSAPEG-Biot.(PEG:PSA, 12:1) PSAPEG-Biot.(PEG:PSA, 24:1) PSAPEG-Biot.(PEG:PSA, 6:1)
PSAunmodified
2D Epoxy
2D Alkyne
0 5000 10000 15000 20000
FU
PSAunmodified PSA-N3
PSA
unmodified
PEG :PSA
, 6:1 PEG
:PSA , 12:1 PEG
:PSA , 24:1
PSA -N3 0
5000 10000 15000
FU
PSAPEG-Biot.
PSA
-N3 w/o Cu PSA
-N3 Cuhigh PSA
-N3 Cu
low 0
5000 10000 15000
FU
Figure 2.8: Immobilization strategies data
A - Immobilization yield of azide-functionalized PSA on 2D-alkyne surface.
Copper concentration: high = 100µM, low = 100 nM.
B - Immobilization yield of unmodified and azide-functionalized PSA on different surfaces. PSA-N3 did not bind on the epoxy surface because free amine residues were blocked by the modification with NHS-PEG4-N3. C- SEC chromatogram of PEGylated PSA. D - Immobilization yield of functionalized PSA on streptavidin surface
Immobilization with spacer
Apart from an oriented immobilization, it was further investigated whether the detection could be increased if PSA was linked to the surface via a single spacer. In this way, the protein would be able to float in the sample solution being more accessible for antibodies.
The immobilization via a spacer demanded for a conjugation with a bifunctional PEG featuring an amine reactive N-Hydroxysuccinimide (NHS) end for the linkage with PSA as well as a biotin end to connect it to a streptavidin-coated surface. In this regard, the PEGylation degree had to be adjusted to ensure a mono-PEGylation as it was assumed that, due to sterical hindrance, a di-PEGylation would block the antibody from binding.
The PEGylated PSA could be detected and purified employing size exclusion chromatog-raphy (SEC), as seen in figure 2.8 C. In additional MALDI experiments (data not shown), we could further confirm that PSA was preferably mono-PEGylated (≈33 kDa) if a molar PEG:PSA ratio of 6:1 was used for conjugation. An increased amount of di-PEGylated PSA (≈ 39 kDa) was detected at a molar PEG:PSA ratio of 12:1 and di- as well as tri-PEGylated PSA was generated with a molar ratio of 24:1.
Based on the results represented in figure 2.8 D, the detectability of PSA could not be increased if anchored to the surface via a 5 kDa PEG spacer. Moreover, the highest recog-nition was achieved by unmodified PSA, which unspecifically attached to the streptavidin surface. An explanation might be that the PEG spacer is not rigid enough to hold the pro-tein in an upright position. In this case, PSA might lay down covered by other PEG chains comparable with rolled lumber. To confirm this assumption, additional experiments us-ing time-of-flight secondary ion mass spectrometry (Tof-SIMS) or atomic force microscopy would have to be carried out [18, 19].
Table 2.2: Summarized comparison of different immobilization strategies.
Immobilization Background Modification Modification Detectability P
Strategy required yield [+]
2D Alkyne +++ y ++ o 5
3D Alkyne ++ y ++ o 4
2D Epoxy ++ n +++ +++ 8
3D Epoxy o n +++ ++ 5
3D Malemide o n +++ ++ 4
Neutravidin + y + + 2
Streptavidin + y + + 2
Comparing the acquired information on background intensity, modification performance and achieved detectability for each immobilization strategy, as done in table 2.2, we could conclude that strategies, which did not require a PSA modification step, exhibited a better performance. This could also be considered as time and material saving advantage, when thinking of a multiplexed analysis with many protein candidates. The 2D epoxy surface
was finally chosen as the best protein detectability could be achieved with unmodified PSA here and as it provided acceptable background properties.